EP1474119B1 - Use of extracts containing phytoestrogen selectively modulating estrogen-receptor-beta - Google Patents

Use of extracts containing phytoestrogen selectively modulating estrogen-receptor-beta Download PDF

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EP1474119B1
EP1474119B1 EP03706473A EP03706473A EP1474119B1 EP 1474119 B1 EP1474119 B1 EP 1474119B1 EP 03706473 A EP03706473 A EP 03706473A EP 03706473 A EP03706473 A EP 03706473A EP 1474119 B1 EP1474119 B1 EP 1474119B1
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beta
extracts
vac
estrogen receptor
receptor
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EP1474119A1 (en
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Wolfgang Wuttke
Hubertus Jarry
Volker Christoffel
Barbara Spengler
Michael Popp
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Bionorica AG
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    • A61P9/12—Antihypertensives

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  • the present invention relates to the use of phytoestrogen ambiencen extracts which selectively modulate the estrogen receptor beta [ER-beta] without having a uterotropic effect according to the preamble of claim 1.
  • Extracts from Vitex Agnus Castus have been used for some time in the treatment of control rate abnormalities, mastodynia, premenstrual syndrome, menstrual disorders (due to primary and secondary luteal insufficiency), however such extracts have not been used for estrogen replacement therapy.
  • estradiol levels fall as a result of the disappearance of ovarian function. This results in a weakening of proliferative processes and leads to an increase in the activity of the GnRH pulse generator in the hypothalamus.
  • the gonadotropin releasing hormone pulse generator is a type of clock in the hypothalamus that clocks the pulsatile release of LH, with steroids affecting amplitude and frequency.
  • the resulting stimulated release of LH in the menopausal woman leads to disturbing upward flushes, the so-called "hot flushes".
  • the extracts for use according to the invention can be used in pharmaceutically conventional form, for example in the form of drops, tablets, film-coated tablets, capsules, granules, dragees, suppositories, ointment creams or the like.
  • Extracts of Vitex Agnus Castus are according to the invention, obtainable by ethanolic extraction of the plant or the plant parts.
  • the inventors have used a competitive estrogen receptor assay in which radiolabelled estradiol from the receptors is liganded by ligands that also bind to the receptor, hereafter called receptor modulators; is displaced.
  • receptor modulators radiolabelled estradiol from the receptors
  • non-radioactive estradiol and on the other hand the extract to be investigated were used as receptor modulators.
  • the receptor preparation consisted of the cytosolic fraction from porcine uteri, which contains both estrogen receptor subtypes, namely ER-alpha and ER-beta. The results of a typical experiment are in Fig. 1 shown.
  • FIG. 1 shows the displacement curves of estradiol (E 2 , open symbols) and of VAC extracts (dark symbols).
  • the ordinate indicates the percentage of the (remaining) binding of radioactively labeled estradiol to the estrogen receptor.
  • the upper abscissa denotes the E 2 concentration, while the lower abscissa represents the VAC concentration.
  • the displacement obtained with the lowest concentration of the test substances becomes defined as 100%.
  • Mean ⁇ SEM were obtained from a triplicate.
  • the EC 50 of VAC is 7.8 ⁇ g / ml
  • FIG. 2 Figure 8 shows the displacement curves of radioactively labeled estradiol from human endometrial ER by estradiol (E 2 , open symbols) and VAC extracts (dark symbols).
  • the ordinate indicates the percentage of the (remaining) binding of radioactively labeled estradiol to the estrogen receptor.
  • the upper abscissa denotes the E 2 concentration, while the lower abscissa represents the VAC concentration.
  • the displacement obtained with the lowest concentration of the test substances is defined as 100%.
  • Mean ⁇ SEM were obtained from a triplicate.
  • the EC 50 of VAC is 12.7 ⁇ g / ml.
  • ER-alpha or ER-beta subtype specific ER assays
  • Fig. 3 The result in the form of a typical ER-alpha displacement curve is in Fig. 3 shown.
  • FIG. 3 shows dose-response curves of estradiol (E 2 , open symbols) and Applicant's VAC extracts (dark symbols) in an assay with recombinant human ER-alpha.
  • the upper abscissa indicates the E 2 concentration, while the lower one indicates the VAC concentration.
  • the binding obtained with the lowest concentration of the test substances was defined as 100%.
  • Means ⁇ SEM were obtained from triplicate determinations per concentration value.
  • FIG. 4 shows dose-response curves of estradiol (E 2 , open symbols) and Applicant's VAC extracts (dark symbols) in an assay with recombinant human ER-beta.
  • the upper abscissa indicates the E 2 concentration, while the lower one indicates the VAC concentration.
  • the displacement obtained with the lowest concentration of the test compounds was defined as 100%.
  • Means ⁇ SEM were obtained from triplicate determinations per concentration value.
  • the EC 50 of VAC is 9.9 ⁇ g / ml.
  • Fig. 5 The uterine weights of the rats are plotted on the ordinate.
  • the individual bars concern the control group (only ovariectomized), an estradiol positive control and the effects with two different VAC extract concentrations.
  • a uterotropic effect as estradiol could according to Fig. 5 in the VAC-treated animals, this means that VAC does not proliferatively affect the uterine tissue.
  • VAC can exert estrogenic effects on other organ systems.
  • Fig. 6 shows the effect of estradiol and two different concentrations of VAC on the paratibial adipose tissue, represented as the percentage area of adipose tissue of a CT scan in the area of the tibia compared to a control.
  • Fig. 7 shows the effect of estradiol and two different VAC concentrations on serum leptin content, plotted on the ordinate in ng / ml.
  • Fig. 8 is the maximum intra-bladder pressure in mmHg on the ordinate measured for one control, estradiol as a positive control, and two different VAC concentrations.
  • Fig. 9 shows the area under the pressure measurement curve [AUC] in mmHg x sec for one control, estradiol as positive control and two different VAC concentrations.

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Abstract

The present invention relates to the use of phytoestrogen-containing extracts that selectively modulate estrogen receptor beta (ER-beta) without producing any uterotropic effect, for the treatment of clinical situations and pathophysiological conditions that are selected from the group consisting of: Obesity and thereby to possibly influence the metabolic syndrome, particularly hypertension, asterosclerosis, cardiac infarct, hyperandrogenemia; Menopausal continence disorders; Menopausal heat surges associated with hyperstimulation of the hypothalamic gonadotropin releasing hormone pulse generator; steroid hormonal synthesis disorder, particularly that of progesterone synthesis of the human corpus luteum, resulting in luteal insuffeciency; Alzheimer's disease associated with elevated expression of estrogen receptor beta in hippocampal neurons.

Description

Die vorliegende Erfindung betrifft die Verwendung von phytoestrogenhaltigen Extrakten, die selektiv den Estrogen-Rezeptor-beta [ER-beta] modulieren ohne eine uterotrope Wirkung aufzuweisen gemäß dem Oberbegriff des Anspruchs 1.The present invention relates to the use of phytoestrogenhaltigen extracts which selectively modulate the estrogen receptor beta [ER-beta] without having a uterotropic effect according to the preamble of claim 1.

Extrakte aus Vitex Agnus Castus (Mönchspfeffer, Keuschlamm) sind seit längerer Zeit zur Behandlung von Regeltempoanomalien, Mastodynie, prämenstruellem Syndrom, Störungen der Regelblutung (infolge primärer u. sekundärer Gelbkörperinsuffizienz) im Gebrauch, jedoch wurden derartige Extrakte nicht zur Estrogenersatztherapie eingesetzt.Extracts from Vitex Agnus Castus (Chaste Tree, Chaste Lamb) have been used for some time in the treatment of control rate abnormalities, mastodynia, premenstrual syndrome, menstrual disorders (due to primary and secondary luteal insufficiency), however such extracts have not been used for estrogen replacement therapy.

In der Menopause kommt es zu einem Absinken der Estradiolspiegel in Folge des Erlöschens der Ovarialfunktion. Dies resultiert in einer Abschwächung proliferativer Prozesse und führt im Hypothalamus zu einer Verstärkung der Aktivität des GnRH-Pulsgenerators. (Der Gonadotropin-Releasing-Hormon-Pulsgenerator ist eine Art Taktgeber im Hypothalamus und taktet die pulsatile Ausschüttung von LH, wobei Steroide die Amplitude und die Frequenz beeinflussen.) Die resultierende stimulierte LH-Ausschüttung führt bei der klimakterischen Frau zu störend empfundenen aufsteigenden Hitzwallungen, den sogenannten "Hot flushes".At menopause, estradiol levels fall as a result of the disappearance of ovarian function. This results in a weakening of proliferative processes and leads to an increase in the activity of the GnRH pulse generator in the hypothalamus. (The gonadotropin releasing hormone pulse generator is a type of clock in the hypothalamus that clocks the pulsatile release of LH, with steroids affecting amplitude and frequency.) The resulting stimulated release of LH in the menopausal woman leads to disturbing upward flushes, the so-called "hot flushes".

In Abwesenheit genügend hoher Estradiolspiegel im Blut überwiegt im Knochengewebe die Aktivität der Osteoklasten und damit der Abbau der Knochenmasse, der mit erhöhter Bruchgefahr des Skelettes einhergeht. Gleichzeitig besteht langfristig die Gefahr der Plaquebildung im Gefäßsystem und damit das erhöhte Risiko von Infarkten.In the absence of sufficiently high estradiol levels in the blood, the activity of the osteoclasts and thus the degradation of the bone mass, which is accompanied by an increased risk of fracture of the skeleton, predominates in the bone tissue. At the same time there is a long-term risk of plaque formation in the vascular system and thus the increased risk of infarction.

Nachteilig wären estrogenartige Wirkungen auf Uterus, Vagina, Brustgewebe und Leber. Unerwünscht hieran ist jedoch, dass bislang kein Phytopharmakon im Stand der Technik zur Verfügung stand, welches zu einer organselektiven Prophylaxe oder Therapie bei Estrogenmangel verwendet werden kann.A disadvantage would be estrogen-like effects on the uterus, vagina, breast tissue and liver. Unwanted, however, is that so far no phytopharmaceutical was available in the prior art, which can be used for an organ-selective prophylaxis or therapy in estrogen deficiency.

Dokument WO 01/74345 A2 beschreibt eine Reihe von Leguminosen als Phytoestrogenquelle zur Behandlung von Fettleibigkeit.document WO 01/74345 A2 describes a number of legumes as a source of phytoestrogen for the treatment of obesity.

Ausgehend von diesem Stand der Technik ist es daher Aufgabe der vorliegenden Erfindung, pflanzliche Arzneimittel mit estrogenartiger Wirkung zur Verfügung zu stellen.Based on this prior art, it is therefore an object of the present invention to provide herbal medicines with estrogen-like action available.

Die Lösung dieser Aufgabe erfolgt durch eine Verwendung gemäß Anspruch 1.The solution of this object is achieved by a use according to claim 1.

Mit den phytoestrogenhaltigen Extrakten, insbesondere aus Vitex Agnus castus, vorzugsweise Früchten, der vorliegenden Erfindung ist es erstmals möglich, eine selektive ER-beta-Wirkung - ohne uterotrope Wirkung - zu erzielen. Durch die verwendeten Vitex Agnus castus Extrakte ist es durch Modulation des ER-beta möglich, die folgenden Krankheitsbilder und pathophysiologischen Zustände zu behandeln:

  • Fettleibigkeit und dadurch mögliche Beeinflussung des metabolischen Syndroms, insbesondere Hypertonus, Arteriosklerose, Herzinfarkt, Hyperandrogenämie
With the phytoestrogenhaltigen extracts, in particular from Vitex Agnus castus, preferably fruits, the present invention, it is now possible to achieve a selective ER-beta effect - without uterotropic effect. Through the use of Vitex Agnus castus extracts it is possible by modulation of ER-beta to treat the following conditions and pathophysiological conditions:
  • Obesity and thus possible influence on the metabolic syndrome, in particular hypertension, arteriosclerosis, myocardial infarction, hyperandrogenemia

Die Extrakte zur erfindungsgemäßen Verwendung können in pharmazeutisch üblicher Form verwendet werden, beispielsweise in Form von Tropfen, Tabletten, Filmtabletten, Kapseln, Granulaten, Dragees, Suppositorien, Salben Cremes oder dgl..The extracts for use according to the invention can be used in pharmaceutically conventional form, for example in the form of drops, tablets, film-coated tablets, capsules, granules, dragees, suppositories, ointment creams or the like.

Extrakte aus Vitex Agnus Castus, insbesondere deren Früchte und/oder Blätter sind erfindungsgemäß, erhältlich durch ethanolische Extraktion der Pflanze oder den Pflanzenteilen.Extracts of Vitex Agnus Castus, in particular their fruits and / or leaves are according to the invention, obtainable by ethanolic extraction of the plant or the plant parts.

Um das Vorliegen von Phytoestrogenen in Vitex Agnus castus (VAC) - Extrakten zu zeigen, haben die Erfinder einen kompetitiven Estrogenrezeptor-Assay verwendet, bei welchem radioaktiv markiertes Estradiol von den Rezeptoren durch Liganden, die ebenfalls an den Rezeptor binden, im folgenden Rezeptormodulatoren genannt; verdrängt wird. Als Rezeptormodulatoren wurden einerseits nichtradioaktives Estradiol und andererseits der zu untersuchende Extrakt eingesetzt. Die Rezeptorpräparation bestand aus der cytosolischen Fraktion aus Schweineuteri, welcher beide Estrogenrezeptor-Subtypen, nämlich ER-alpha und ER-beta enthält. Die Ergebnisse eines typischen Experiments sind in Fig. 1 gezeigt.To demonstrate the presence of phytoestrogens in Vitex Agnus castus (VAC) extracts, the inventors have used a competitive estrogen receptor assay in which radiolabelled estradiol from the receptors is liganded by ligands that also bind to the receptor, hereafter called receptor modulators; is displaced. On the one hand non-radioactive estradiol and on the other hand the extract to be investigated were used as receptor modulators. The receptor preparation consisted of the cytosolic fraction from porcine uteri, which contains both estrogen receptor subtypes, namely ER-alpha and ER-beta. The results of a typical experiment are in Fig. 1 shown.

Figur 1 zeigt die Verdrängungskurven von Estradiol (E2, offene Symbole) und von VAC-Extrakten (dunkle Symbole). Die Ordinate gibt den prozentualen Anteil der (noch verbleibenden) Bindung von radioaktiv markiertem Estradiol an den Estrogenrezeptor an. Die obere Abszisse bezeichnet die E2-Konzentration, während die untere Abszisse die VAC-Konzentration wiedergibt. Diejenige Verdrängung, welche mit der niedrigsten Konzentration der Testsubstanzen erhalten wurde, wird als 100% definiert. Mittelwerte ± SEM wurden aus einer Dreifachbestimmung erhalten. Die EC50 von VAC beträgt 7.8 µg/ml FIG. 1 shows the displacement curves of estradiol (E 2 , open symbols) and of VAC extracts (dark symbols). The ordinate indicates the percentage of the (remaining) binding of radioactively labeled estradiol to the estrogen receptor. The upper abscissa denotes the E 2 concentration, while the lower abscissa represents the VAC concentration. The displacement obtained with the lowest concentration of the test substances becomes defined as 100%. Mean ± SEM were obtained from a triplicate. The EC 50 of VAC is 7.8 μg / ml

Wie aus Fig. 1 ersichtlich, konkurrieren die Verbindungen in den VAC-Extrakten dosisabhängig mit radioaktiv markiertem Estradiol um die Bindungsstellen der Estrogenrezeptoren . Die erhaltenen Daten demonstrieren eindrucksvoll, daß die untersuchten Extrakte Phytoestrogene enthalten.How out Fig. 1 As can be seen, the compounds in the VAC extracts dose-dependently compete with radioactively labeled estradiol for the binding sites of the estrogen receptors. The obtained data demonstrate impressively that the examined extracts contain phytoestrogens.

Um diesen Befund noch stärker zu untermauern, wurde der gleiche Assay mit einer Rezeptor-Präparation aus humanem Endometrium durchgeführt. Wiederum liegen beide ER-Subtypen in der Lösung vor. Die Ergebnisse dieser Untersuchungen sind in Fig. 2 gezeigt.To further support this finding, the same assay was performed with a receptor preparation from human endometrium. Again, both ER subtypes are present in the solution. The results of these investigations are in Fig. 2 shown.

Figur 2 zeigt die Verdrängungskurven von radioaktiv markiertem Estradiol von ER aus humanem Endometrium durch Estradiol (E2, offene Symbole) und VAC-Extrakte (dunkle Symbole) . Die Ordinate gibt den prozentualen Anteil der (noch verbleibenden) Bindung von radioaktiv markiertem Estradiol an den Estrogenrezeptor an. Die obere Abszisse bezeichnet die E2-Konzentration, während die untere Abszisse die VAC-Konzentration wiedergibt. Diejenige Verdrängung, welche mit der niedrigsten Konzentration der Testsubstanzen erhalten wurde, wird als 100% definiert. Mittelwerte ± SEM wurden aus einer Dreifachbestimmung erhalten. Die EC50 von VAC beträgt 12,7 µg/ml. FIG. 2 Figure 8 shows the displacement curves of radioactively labeled estradiol from human endometrial ER by estradiol (E 2 , open symbols) and VAC extracts (dark symbols). The ordinate indicates the percentage of the (remaining) binding of radioactively labeled estradiol to the estrogen receptor. The upper abscissa denotes the E 2 concentration, while the lower abscissa represents the VAC concentration. The displacement obtained with the lowest concentration of the test substances is defined as 100%. Mean ± SEM were obtained from a triplicate. The EC 50 of VAC is 12.7 μg / ml.

Die Ergebnisse zeigen klar, daß Phytoestrogene aus VAC-Extrakten an die humanen Estrogenrezeptoren binden, was das molekulare Initialereignis bei einer estrogenartigen Wirkung von Verbindungen aus VAC beim Menschen ist.The results clearly show that phytoestrogens from VAC extracts bind to the human estrogen receptors, which is the initial molecular event in an estrogen-like effect of compounds from VAC in humans.

Aufgrund der Verfügbarkeit von rekombinanten Rezeptorproteinen können, subtypspezifische ER Assays (ER-alpha oder ER-beta) durchgeführt werden. Das Ergebnis in Form einer typischen ER-alpha-Verdrängungskurve ist in Fig. 3 gezeigt.Due to the availability of recombinant receptor proteins, subtype specific ER assays (ER-alpha or ER-beta) can be performed. The result in the form of a typical ER-alpha displacement curve is in Fig. 3 shown.

Die Figur 3 zeigt Dosis/Wirkungskurven von Estradiol (E2, offene Symbole) und den VAC Extrakten der Anmelderin (dunkle Symbole) in einem Assay mit rekombinantem humanen ER-alpha. Die obere Abszisse gibt die E2 - Konzentration an, während die untere die VAC - Konzentration angibt. Diejenige Bindung, welche mit der niedrigsten Konzentration der Testsubstanzen erhalten wurde, wurde mit 100% definiert. Mittelwerte ± SEM wurden aus Dreifachbestimmungen pro Konzentrationswert erhalten.The FIG. 3 shows dose-response curves of estradiol (E 2 , open symbols) and Applicant's VAC extracts (dark symbols) in an assay with recombinant human ER-alpha. The upper abscissa indicates the E 2 concentration, while the lower one indicates the VAC concentration. The binding obtained with the lowest concentration of the test substances was defined as 100%. Means ± SEM were obtained from triplicate determinations per concentration value.

Während E2 eine dosisbezogene Konkurrenz zu dem radioaktiven Rezeptorliganden zeigt, binden die Phytoestrogene des VAC-Extraktes nicht an den ER-alpha. Somit konnte keine EC50 berechnet werden.While E 2 shows dose-related competition with the radioactive receptor ligand, the phytoestrogens of the VAC extract do not bind to the ER-alpha. Thus, no EC 50 could be calculated.

Mit denselben Testverbindungen wie beim ER-alpha-Assay, wurden nun ER-beta-Assays durchgeführt. Die Daten und der Graph eines repräsentativen Experimentes sind in Fig. 4 gezeigt.With the same test compounds as in the ER-alpha assay, ER-beta assays have now been performed. The data and graph of a representative experiment are in Fig. 4 shown.

Die Figur 4 zeigt Dosis/Wirkungskurven von Estradiol (E2, offene Symbole) und den VAC Extrakten der Anmelderin (dunkle Symbole) in einem Assay mit rekombinantem humanen ER-beta. Die obere Abszisse gibt die E2 - Konzentration an, während die untere die VAC - Konzentration angibt. Diejenige Verdrängung, welche mit der niedrigsten Konzentration der Testverbindungen erhalten wurde, wurde als 100% definiert. Mittelwerte ± SEM wurden aus Dreifachbestimmungen pro Konzentrationswert erhalten. Die EC50 von VAC beträgt 9,9 µg/ml.The FIG. 4 shows dose-response curves of estradiol (E 2 , open symbols) and Applicant's VAC extracts (dark symbols) in an assay with recombinant human ER-beta. The upper abscissa indicates the E 2 concentration, while the lower one indicates the VAC concentration. The displacement obtained with the lowest concentration of the test compounds was defined as 100%. Means ± SEM were obtained from triplicate determinations per concentration value. The EC 50 of VAC is 9.9 μg / ml.

Im Gegensatz zum ER-alpha-Assay binden die Phytoestrogene der VAC-Extrakte der Anmelderin in dosisabhängiger Weise an den ER-beta. Diese Daten bestätigen ohne jeden Zweifel die ER-beta-Selektivität der Vitex Agnus castus Extrakte der Anmelderin.In contrast to the ER-alpha assay, the phytoestrogens of Applicant's VAC extracts bind in a dose-dependent manner to the ER-beta. These data unequivocally confirm the ER-beta selectivity of the Applicant's Vitex Agnus castus extracts.

Zur Überprüfung des uterotropen Effektes wurde folgendes Experiment durchgeführt, dessen Daten graphisch in Fig. 5 gezeigt sind.To check the uterotropic effect, the following experiment was carried out, whose data are plotted in Fig. 5 are shown.

Über drei Monate wurden zwei unterschiedliche Konzentration von VAC-Extrakten (100mg/kg = VAC100 und 400mg/kg Körpergewicht = VAC400) dem Futter von ovariektomierten Rattenweibchen und orchidektomierten Männchen beigemischt. VAC hatte im Gegensatz zu Estradiol keinen uterotropen Einfluß und auch das Vaginalepithel blieb unbeeinflußt. Beide proliferativen Effekte des Estradiols sind im Rahmen der peri- und postklimatkterischen Estrogenersatztherapie bzw. einer Hormonersatztherapie bei Frauen nach einer Ovariektomie unerwünscht, da bei ständiger Gabe von Östrogenen und fehlender Gegenregulation durch Gestagene eine Hyperplasie des Endometriums durch den andauernden proliferativen Effekt des Estradiols nicht ausgeschlossen werden kann.Over three months, two different concentrations of VAC extracts (100 mg / kg = VAC100 and 400 mg / kg body weight = VAC400) were added to the diet of ovariectomized female rats and orchidectomized males. In contrast to estradiol, VAC had no uterotropic influence and the vaginal epithelium was unaffected. Both proliferative effects of estradiol are in the context of peri- and postklimatkterischen estrogen replacement therapy or hormone replacement therapy in women after ovariectomy undesirable because with constant administration of estrogens and lack of counterregulation by progestins hyperplasia of the endometrium are not excluded by the ongoing proliferative effect of estradiol can.

In Fig. 5 sind auf der Ordinate die Uterusgewichte der Ratten aufgetragen. Die einzelnen Balken betreffen die Kontrollgruppe (nur ovariektomiert), eine Estradiol-Positivkontrolle sowie die Effekte mit zwei unterschiedlichen VAC-Extraktkonzentrationen.In Fig. 5 The uterine weights of the rats are plotted on the ordinate. The individual bars concern the control group (only ovariectomized), an estradiol positive control and the effects with two different VAC extract concentrations.

Eine uterotrope Wirkung wie beim Estradiol konnte gemäß Fig. 5 bei den mit VAC behandelten Tieren nicht festgestellt werden, das bedeutet, daß VAC nicht proliferativ auf das Uterusgewebe wirkt.A uterotropic effect as estradiol could according to Fig. 5 in the VAC-treated animals, this means that VAC does not proliferatively affect the uterine tissue.

Jedoch wurde bei weiteren Untersuchungen überraschend gefunden, daß VAC, estrogene Effekte auf andere Organsysteme ausüben kann.However, in further studies it has surprisingly been found that VAC can exert estrogenic effects on other organ systems.

Mit computerassistierter Tomographie wurde das abdominale und paratibiale Fettgewebe bei männlichen und weiblichen Ratten bestimmt. Unter E2 und der hohen Dosis von Vitex Agnus castus ist eine Reduktion beider Gewebsanteile beobachtet worden. Gleichzeitig sind die Serum-Leptinspiegel der VAC behandelten Tiere abgesenkt. Die Ergebnisse dieser Untersuchung sind in den Figuren 6 und 7 gezeigt.Computer-assisted tomography was used to determine abdominal and paratibial adipose tissue in male and female rats. Under E2 and the high dose of Vitex Agnus castus, a reduction in both tissue levels has been observed. At the same time, serum leptin levels of VAC-treated animals are lowered. The results of this study are in the FIGS. 6 and 7 shown.

Fig. 6 zeigt die Wirkung von Estradiol und zwei unterschiedlichen VAC-Konzentrationen auf das paratibiale Fettgewebe, dargestellt als prozentualer Flächenanteil des Fettgewebes einer CT-Aufnahme im Bereich der Tibia gegenüber einer Kontrolle. Fig. 6 shows the effect of estradiol and two different concentrations of VAC on the paratibial adipose tissue, represented as the percentage area of adipose tissue of a CT scan in the area of the tibia compared to a control.

Fig. 7 zeigt die Wirkung von Estradiol und zwei unterschiedlichen VAC-Konzentrationen auf den Serumleptingehalt, auf der Ordinate aufgetragen in ng/ml. Fig. 7 shows the effect of estradiol and two different VAC concentrations on serum leptin content, plotted on the ordinate in ng / ml.

Wie aus den Figuren 6 und 7 ersichtlich, zeigt sich eine Senkung des paratibialen Fettgehaltes und eine Senkung der Serumleptinkonzentration unter VAC-Gabe.Like from the FIGS. 6 and 7 As can be seen, a reduction in the paratibial fat content and a decrease in serum leptin concentration under VAC administration.

Ebenfalls mit computerassistierter Tomographie wurde die Veränderung der Knochendichte (geringere Osteoporoseentwicklung) an der Metaphyse der Tibia bei Rattenmännchen und -weibchen beobachtet. Es zeigte sich, daß unter VAC die Knochendichte deutlich weniger abnimmt, als bei den unbehandelten Kontrolltieren.Also, with computer-assisted tomography, the change in bone density (lower osteoporosis development) in the metaphysis of the tibia was observed in rat males and females. It was found that under VAC the bone density decreases significantly less than in the untreated control animals.

Die Instillation von 1 ml physiologischer Kochsalzlösung mit einem Katheter in die Blase weiblicher ovariektomierter Ratten innerhalb von 1 min führt zu einem Anstieg des Blaseninnendruckes. Bei Tieren die drei Monate lang mit E2-substituiert waren fällt der Druckanstieg etwa dreifach höher aus als bei den scheinbehandelten Kontrolltieren und ist geringfügig schwächer aber signifikant als bei den Estradiolbehandelten Tieren, wenn die Tiere mit VAC behandelt wurden. Die Ergebnisse dieser Untersuchung sind in den Figuren 8 und 9 gezeigt.The instillation of 1 ml of physiological saline with a catheter into the bladder of female ovariectomized rats within 1 min leads to an increase in the internal bladder pressure. In animals that were E2-substituted for three months, the pressure increase is about three-fold higher than the sham-treated control animals and is slightly weaker but significantly lower than in the estradiol-treated animals when treated with VAC. The results of this study are in the FIGS. 8 and 9 shown.

In Fig. 8 ist der maximale Blaseninnendruck in mmHg auf der Ordinate angegeben, der für eine Kontrolle, Estradiol als Positivkontrolle und zwei unterschiedliche VAC-Konzentrationen gemessen wurde.In Fig. 8 is the maximum intra-bladder pressure in mmHg on the ordinate measured for one control, estradiol as a positive control, and two different VAC concentrations.

Fig. 9 zeigt dagegen auf der Ordinate die Fläche unter der Druckmeßkurve [AUC] in mmHg x sec für eine Kontrolle, Estradiol als Positivkontrolle und zwei unterschiedliche VAC-Konzentrationen. Fig. 9 on the other hand, the ordinate shows the area under the pressure measurement curve [AUC] in mmHg x sec for one control, estradiol as positive control and two different VAC concentrations.

Nach alledem ist es somit möglich, VAC-Extrakte zur Herstellung von Arzneimitteln zur Behandlung von:

  • Fettleibigkeit und dadurch einer möglichen Beeinflussung des metabolischen Syndroms, insbesondere Hypertonus, Arteriosklerose, Herzinfarkt, Hyperandrogenämie ohne uterotrope Wirkung einzusetzen.
After all, it is thus possible to use VAC extracts for the preparation of medicaments for the treatment of:
  • Obesity and thus a possible influence on the metabolic syndrome, in particular hypertension, arteriosclerosis, myocardial infarction, use hyperandrogenemia without uterotropic effect.

Literatur:Literature:

  1. 1.) Hrabovszky E, Shughrue PJ, Merchenthaler I, Hajszan T, Carpenter CD, Liposits Z, Petersen SL Detection of estrogen receptor-beta messenger ribonucleic acid and 125l-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain. Endocrinology 2000 Sep; 141 (9):3506-9 1.) Hrabovszky E, Shughrue PJ, Merchenthaler I, Hajszan T, Carpenter CD, Liposits Z, Petersen SL Detection of estrogen receptor-beta messenger ribonucleic acid and 125l-estrogen binding sites in luteinizing hormone-releasing hormones neurons of the rat brain. Endocrinology 2000 Sep; 141 (9): 3506-9
  2. 2.) Hosokawa K, Ottander U, Wahlberg P, Ny T, Cajander S, Olofsson IJ. Dominant expression and distribution of oestrogen receptor beta over oestrogen receptor alpha in the human corpus luteum. Mol Hum, Reprod 2001 Feb;7(2):137-45 2.) Hosokawa K, Ottander U, Wahlberg P, Ny T, Cajander S, Olofsson IJ. Dominant expression and distribution of estrogen receptor beta in oestrogen receptor alpha in the human corpus luteum. Mol Hum, Reprod 2001 Feb; 7 (2): 137-45
  3. 3.) Taylor AH, Al-Azzawi F. Immunolocalisation of oestrogen receptor beta in human tissues. J Mol Endocrinol 2000 Feb;24(1):145-55 3.) Taylor AH, Al-Azzawi F. Immunolocalization of estrogen receptor beta in human tissues. J Mol Endocrinol 2000 Feb; 24 (1): 145-55
  4. 4.) Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138:863-870 4.) Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptor alpha and beta. Endocrinology 138: 863-870
  5. 5.) Saunders PT, Maguire SM, Gaughan J, Millar MR, 1997 Expression of oestrogen receptor beta (ER beta) in multiple rat tissues visualised by immunohistochemistry. J Endocrinol 154:R13-R16 5.) Saunders PT, Maguire SM, Gaughan J, Millar MR, 1997 Expression of estrogen receptor beta (ER beta) in multiple rat tissues visualized by immunohistochemistry. J Endocrinol 154: R13-R16
  6. 6.) Mäkelä S, Strauss L, Kuiper G, Valve E, Salmi S, Santti R, Gustafsson JA, 2000 Differential expression of estrogen receptors alpha and beta in adult rat accessory sex glands and lower urinary tract. Mol Cell Endocrinol 164:109-116 6.) Mäkelä S, Strauss L, Kuiper G, Valve E, Salmi S, Santti R, Gustafsson JA, 2000 Differential expression of estrogen receptor alpha and beta in adult rat sex glands and lower urinary tract. Mol Cell Endocrinol 164: 109-116

Claims (4)

  1. Use of phytoestrogen-containing extracts exclusively from Vitex agnus castus, for preparing a medicament for selectively modulating the estrogen receptor beta without producing a uterotropic effect, characterized in that the selective modulation of the estrogen receptor beta is used in the treatment of clinical situations and pathophysiological conditions of obesity and an influence on the metabolic syndrome thereby possible, particularly hypertension, arteriosclerosis, cardiac infarct, hyperandrogenemia, said extracts being obtainable by ethanol extraction.
  2. Use according to claim 1, characterized in that the extract is used in a standard pharmaceutical formulation.
  3. Use according to claim 2, characterized in that drops, tablets, coated tablets, capsules, granulates, dragées, suppositories, ointments, creams are used as pharmaceutical formulations.
  4. Use according to any one of claims 1 to 3, characterized in that the extracts from Vitex agnus castus are obtainable from the fruits and/or leaves thereof.
EP03706473A 2002-02-15 2003-02-10 Use of extracts containing phytoestrogen selectively modulating estrogen-receptor-beta Expired - Lifetime EP1474119B1 (en)

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